WO1999025331A1 - Use of ketoacids together with amino acids for enhancing muscle performance and recovery from fatigue - Google Patents

Use of ketoacids together with amino acids for enhancing muscle performance and recovery from fatigue Download PDF

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Publication number
WO1999025331A1
WO1999025331A1 PCT/US1998/024325 US9824325W WO9925331A1 WO 1999025331 A1 WO1999025331 A1 WO 1999025331A1 US 9824325 W US9824325 W US 9824325W WO 9925331 A1 WO9925331 A1 WO 9925331A1
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composition
acid
muscle
fatigue
gakic
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PCT/US1998/024325
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French (fr)
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Bruce R. Stevens
Michael D. Godfrey
Thomas W. Kaminski
Randy W. Braith
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University Of Florida
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Priority to AU14092/99A priority Critical patent/AU1409299A/en
Publication of WO1999025331A1 publication Critical patent/WO1999025331A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group

Definitions

  • Exhaustive exercise depletes metabolic energy carbon sources and acutely disrupts skeletal muscle nitrogen metabolism in three principle ways.
  • Certain amino acids including branched-chain amino acids, are released from muscle and are deaminated to elevate serum ammonia, oxidized locally as muscle fuel sources, and augment metabolic acidosis.
  • Protein catabolism is initiated (including decreasing the rate of protein synthesis, as well as increasing degradation of non- contractible protein), thereby reducing long-term strength gains.
  • ⁇ -ketoisocaproate is the ketoacid parent chain of Z-leucme (i.e., it is Zleucine without the amino nitrogen), and is the first metabolite in the muscle catabolic pathway of leucme following reversible transammation to glutamate (Legaspi, A.
  • erosion of muscle mass induced by overtraining or injury can be reduced by metabolic intervention (Blomstrand, E. and E.A. Newsholme (1992) "Effect of branched-cham ammo acid supplementation of the exercise-mduced change in aromatic ammo acid concentration in human muscle” Ada Physiol. Scand.46:293-298; and Mo ⁇ , E., M. Hasebe,
  • Ammonia (NH 3 ) arises from the deamination of adenosine monophosphate to inosine monophosphate in the purine nucleotide cycle, as well as from the deamination of branched-chain amino acids.
  • the ability of tissues to re-aminate leucine from supplemental ⁇ -ketoisocaproate has been clinically exploited as a means to treat muscle wasting in acutely traumatized and critically ill patients, while reducing their nitrogen load (Harper, A.E., R.H. Miller, K.P.
  • Enhancing muscle recovery following trauma occurs not simply by administering oral or intravenous leucine alone, but instead it responds to increasing the steady-state concentration of ⁇ -ketoisocaproic acid.
  • This anabolic ketoacid is a major factor in reducing protein catabolism, stimulating muscle synthesis, and sparing glucose oxidation, while stimulating insulin release.
  • ⁇ -ketoisocaproic acid is superior to leucine in this regard in human and rat muscle studies (Buckspan, R., B. Hoxworth, E. Cersosimo, J. Devlin, E. Horton, N. Abrumrad (1986) "Alpha-ketoisocaproate is superior to leucine in sparing glucose utilization in humans" Am. J. Physiol. 25 E648-E653).
  • BCAA-aminotransferase The most critical enzymes are BCAA-aminotransferase, the BCKA dehydrogeneases family, L-leucine dehydrogenase, and 3-methyl-2-oxobutanoate dehydrogenase. Enzyme concentrations of BCAA-aminotransferase are relatively unregulated at fairly steady-state levels in muscle. Therefore, transamination is reversibly catalyzed by BCAA-aminotransferase activity through mass action of available concentrations of ⁇ - ketoisocaproate, L-leucine, L-glutamate, ⁇ -ketoglutarate, and their ancillary metabolites.
  • L-leucine dehydrogenase provides a beneficial pathway that catalyzes the NH 3 amination of ⁇ -ketoisocaproate to yield L-leucine.
  • the enzyme 3-methyl-2-oxobutanoate dehydrogenase can catalyze the decarboxylation of ⁇ -ketoisocaproate, leading to pathways eventually creating acetoacetate.
  • Alpha-ketoisocaproic acid can be hydrolyzed to beta-hydroxy-beta-methylbutyrate via ⁇ -ketoisocaproate dioxygenase.
  • BCKA-dehydrogenase In contrast to unregulated BCAA-aminotransferase, the activity of BCKA-dehydrogenase is highly regulated during exercise. In muscle and liver, BCKA-dehydrogenase is a multienzyme complex that catalyzes the irreversible oxidative decarboxylation of BCKA, as it reduces NAD to NADH. The activity of BCKA-dehydrogenase greatly increases immediately after strenuous exercise, with subsequent return to resting baseline levels by 10 minutes post-exercise (Kasperek, GJ.
  • BCKA-dehydrogenase enzyme activity is regulated by an ATP phosphorylation (inactivation)-dephosphorylation (activation) mechanism
  • ⁇ -ketoisocaproate is a key stimulator of this enzyme complex, whereby it inhibits the ATP -mediated kinase allosteric inactivation of BCKA-dehydrogenase.
  • L-Arginine is considered a "conditionally essential” amino acid that becomes essential under certain metabolic conditions including muscle trauma and injury (Dundrick, P.S. and W.W. Souba (1991) "Amino acids in surgical nutrition” Surg. Clin. N Am. 71:459-476 and
  • L-Arginine is a dibasic amino acid that participates in the urea cycle and other intermediary pathways, notably serving as the starting substrate for biosynthesis of polyamines. Polyamines are essential for protein synthesis, and cell growth and proliferation. Arginine synergetically promotes the nitrogen-retaining effects of ⁇ -KIC.
  • the subject invention concerns methods and compositions for enhancing muscle performance and recovery from fatigue. More specifically, the subject invention concerns a unique metabolic intervention to improve athletic dynamic muscle strength and muscle work, and recovery from acute exhaustive anaerobic strength training exercise.
  • the subject invention concerns the use of a glycine and L- arginine monohydrochloride salt of alpha-ketoisocaproic acid calcium (GAKIC) via oral treatment to a human or animal to enhance muscle performance and recovery from fatigue.
  • GAKIC alpha-ketoisocaproic acid calcium
  • GAKIC treatment increases the ability to sustain athletic muscle force during intense anaerobic muscle exercise
  • GAKIC treatment increases the ability to sustain athletic muscle work during intense anaerobic muscle exercise; and 3) GAKIC increases the overall muscle performance by decreasing muscle absolute fatigue while retarding the rate of muscle fatigue.
  • G.AKIC treatment increases the ability to sustain muscle force (torque) up to at least 28% during intense acute anaerobic dynamic muscle exercise;
  • GAKIC treatment increases the ability to sustain muscle total work (joules) by up to 12% during the early phase of intense anaerobic dynamic muscle exercise;
  • GAKIC increases the overall muscle performance by forestalling muscle fatigue during the early phases of anaerobic exercise.
  • the subject invention also concerns a new testing protocol which is able to objectively test, quantify and provide a reproducible assessment of muscle work, fatigue, and recovery of dynamic muscle function associated with acute anaerobic exercise. This protocol was exploited to discriminate the effects of metabolic interventions on muscle function during training regimes.
  • compositions and methods of the subject invention can be used to treat humans and animals.
  • the materials and methods are used to treat horses.
  • Brief Description of the Drawings Figure 1 shows a brief overview of the whole-body intermediary metabolic relationship among BCAA and BCKA.
  • Figure 2 shows the testing protocol overview.
  • Figure 3 shows a representative computation of the fatigue resistance index (FRI).
  • Figure 4 shows the concentric muscle fatigue resistance enhancement by GAKIC.
  • Figure 5 shows the percent gain in fatigue resistance directly attributable to GAKIC treatment.
  • Figure 6 shows the total work performed during each fatigue set for the concentric phase.
  • FIG. 7 shows the work gained from GAKIC treatment.
  • Figure 8 shows the percent increase in total work gained from GAKIC treatment.
  • the subject invention provides materials and methods that enhance muscle performance under conditions of acute exhaustive anaerobic dynamic exercise.
  • the subject invention can be used to enhance human or animal muscle performance.
  • Animals which can benefit from the subject invention include horses.
  • GAKIC a novel formulation useful according to the subject invention, is safe for humans and animals.
  • GAKIC comprises glycine, L-arginine, and ⁇ -ketoisocaproic acid.
  • GAKIC can be used according to the subject invention as a nutritional supplement to enhance athletic performance.
  • the benefits of GAKIC include at least the following:
  • GAKIC is a safe means to enhance athletic performance, without the harmful side effects or illegality of anabolic steroids, pharmaceuticals or elevated caffeine.
  • GAKIC can be useful to medical patients with sports injuries, as a means to improve exercise tolerance and recovery during rehabilitation.
  • the described exercise and testing protocol using the computerized isokinetic dynamometer is a unique and useful means to quantify/assess muscle force, work, and dynamic fatigue performance during concentric and eccentric contractions under well-controlled reproducible experimental conditions. These conditions are directly extrapolated to the real-world of athletic training and competition.
  • the described exercise and testing protocol using the computerized isokinetic dynamometer is a unique and useful means to quantify/assess muscle function in medical patients with sports injuries, as a means to improve exercise tolerance and recovery during rehabilitation.
  • GAKIC can help improve animal jumping and racing performance.
  • the composition of the subject invention can comprises other cationic or dibasic amino acids.
  • These amino acids may be, for example, omithine, histidine, and/or lysine.
  • glutamate and/or glutamine may be substituted for glycine.
  • other salts of alpha-ketoisocaproic acid may be used in the compositions of the subject invention. These salts may be, for example, the sodium or potassium salts. Alternatively, the free acid may be used. It should be noted that the subject invention is not limited only to the use of alpha-ketoisocaproic acid and its salts. Other related acids and salts may also be used.
  • These other related acids and salts may be, for example, alpha-keto-isovaleric acid, alpha-keto-beta-methylvaleric acid, or pyruvic acid as the free acids or as a salt of calcium, potassium sodium, or as a conjugate.
  • compositions of the subject invention may be administered individually, as a mixture, or as covalent bonds, or other conjugate.
  • alpha-keto- isocaproic acid or alpha-keto-isovaleric acid, alpha-keto-beta-methylvaleric acid, or pyruvic acid can be conjugated with arginine.
  • the subject invention pertains to the use of a ketoacid with a cationic amino acid to improve muscle performance and recovery from fatigue.
  • L-isomers are preferred racemic mixtures may also be utilized.
  • One aspect of the subject invention pertains to a stable tripeptide comprised of glycine, L-arginine, and ⁇ -ketoisocaproate covalently linked by amide bonds.
  • this orally consumed GAKIC precursor is readily hydrolyzed by human pancreatic proteases, yielding the appropriate components of GAKIC in vivo in the intestinal lumen or in vitro.
  • a low calorie beverage is provided.
  • low calorie cranberry juice can be used.
  • the components can be orally administered via capsules.
  • Oral administration of the subject glycine-arginine- ⁇ -ketoisocaproic acid formulation is preferred over the intravenous route, due to the coordinated metabolic communication (Figure 1) between skeletal muscle, liver, kidneys and the splanchnic bed, notably the small intestine. Both the splanchnic and skeletal muscle regions are the body's major sites of branched-chain amino acid metabolism.
  • Ketoacids have been given at over 10 gm per day for up to 10 months (Walser, M., N.D. LaFrance, L. Ward, M.A. Van Duyn (1987) "Progression of chronic renal failure in patients given ketoacids following amino acids. Kidney Int. 32: 123-128).
  • compositions of the subject invention can be administered in an effective dosage which can be readily ascertained by a person skilled in the art having the benefit of the current disclosure.
  • a person skilled in the art would appreciate that the ratios as well as the concentrations of components can be modified in order to achieve the desired effect of enhanced muscle performance and reduced muscle fatigue.
  • One aspect of the subject invention concerns unique protocols and assessment instruments which can be used to quantify the effects of metabolic intervention on muscle force, work, and fatigue associated with acute anaerobic exercise. These measurements standardize the results from each individual subject, thereby permitting meaningful compa ⁇ sons withm the repeated-measures randomized crossover design.
  • isokinetic fatigue is commonly assessed by measu ⁇ ng the number of repetitions a subject can perform before force levels drop below a certain standard percentage of peak torque, comparison of complete exhaustion relative to a maximal static force, or assessment of time to exhaustion (Lewis, S.F.,
  • the current invention provides a novel Fatigue Resistance Index (FRI) which provides a uniform measure of dynamic fatigue resistance with respect to each subject's individual force output.
  • FRI Fatigue Resistance Index
  • the FRI provides a meaningful measure of the dynamic resistance to fatigue by allowing all subjects to perform the same concentric and eccent ⁇ c fatigue protocol of four 35 -repetition high intensity sets of 90°/sec knee extensions, regardless of fatigue rate.
  • the protocol and assessment are in contrast to other methods which have attempted using isokinetic measurements to quantify exercise fatigue or by using bicycle ergometers, free weight trials, vertical leap tests, and treadmill running; these methods are not as sensitive to acute dynamic changes in muscle force.
  • Pre-fatigue and post-fatigue peak torque and total work values were measured in isolated quad ⁇ ceps femo ⁇ s of subjects using a computer-controlled isokinetic dynamometer over a 23 day interval.
  • Du ⁇ ng testing fasted subjects were given oral GAKIC or isocalo ⁇ c carbohydrate over a 45 mm period preceding exercise.
  • systolic and diastolic blood pressure systolic and diastolic blood pressure, body weight, legg mass, and dietary L-leucme, L-arginme, glycine, total protein, total calories, total carbohydrate and caffeine.
  • the mean baseline concentric isokinetic knee extension maximal torque was 240 ⁇ 9 Nm.
  • the subject invention is also useful m extended, continual aerobic phases of intense exercise exceeding 15 mm, but less than 24 hr of continuous exercise, that include both anaerobic glycolytic as well as oxidative metabolism due to the metabolic pathways employing the components of GAKIC.
  • GAKIC can also be used for longitudinal weight-trammg.
  • Subject physical characte ⁇ stics were measured during the randomly assigned carbohydrate and GAKIC treatment pe ⁇ ods Because the crossover design used the exact same subjects for both treatments, subject age (21 ⁇ 2) and height (72 ⁇ 2 inches) were the same.
  • the carbohydrate and GAKIC treatment periods were not significantly different (p > 0.05) with respect to body weight (82.93 vs. 82.74 lbs), limb weight (84.77 vs 86.15 Newtons), or m blood pressure immediately preceding to testing (117/74 vs. 119/73 mm Hg; systohc/diastolic). Treatments.
  • GAKIC glycme-L-argmine- ⁇ -ketoisocaproic acid
  • the Fatigue Resistance Index desc ⁇ bes the maximal exerted force sustained under fatiguing conditions as a percent of maximal force exerted under fresh conditions for each individual over a period of time.
  • FRI is based on the ability to produce concent ⁇ c or eccent ⁇ c torque immediately following maximal exertion over 30 initial repetitions of a 35 repetition set in the presence of metabolic treatment, compared to that same individual's ability to produce force under fresh (baseline) conditions in the absence of treatment.
  • the fixed value of 35 maximal isokinetic concent ⁇ c/eccent ⁇ c repetitions to induce fatigue in the quadriceps muscle groups was determined based on a pilot study. A pilot study was initially employed to determine isokinetic fatigue conditions and muscle performance quantifications. Based on this pilot, fatigue was induced m the quad ⁇ ceps muscle group du ⁇ ng a fixed value of 35 maximal isokinetic concentric/eccentric repetitions.
  • BIKEMSV Baseline Isokinetic Knee Extension Maximal Strength Value
  • Total Work Total work (TW) values for each 35 repetition set were also collected and de ⁇ ved from the Km-Con 125AP software, and are expressed m units of joules produced du ⁇ ng each set.
  • Isokinetic Dynamometer Instrumentation Each subject's right knee was carefully aligned with the rotation axis of the dynamometer lever arm, and subjects were secured in place using a waist strap, two should straps, and a thigh stabilizer. The distal shm pad was adjusted for each subject and placed two finger breadths above the lateral malleolus. The shm pad was securely fastened to the level arm using a Velcro strap. Gravity compensation procedures were earned out according to manufactures recommendations with the knee m and extended position.
  • Knee extension and flexion motion occurred in a 75 ° arc, so that start and stop angles were 85 ° and 10°, respectively.
  • TW 4600 ⁇ 280 joules.
  • crossover design i.e., GAKIC or carbohydrate given in either first or second set of trials
  • N 10 for a repeated measures paired t-test.
  • the Expenmental Model Testing effects.
  • a testing protocol on a naive subject, especially one utilizing an uncommon movement type like isokinetic exercise, there is always concern that the results might be affected by a testing or learning effect. This is an improvement m performance that may occur as a result of experience and coordination gained through multiple trials.
  • the double-blind, repeated measures crossover design of this study was invoked to negate such possible testing effects. This design gave each group equal chance to be affected, therefore canceling out any impact of significance; there were no significant (p > 0.05) differences m any treatment attributable to the random order of treatment
  • the Expenmental Model Time variable.
  • the speed at which the GAKIC supplement is absorbed and dist ⁇ ubbed to the skeletal muscle is not completely known, but typical rates of gast ⁇ c emptying and intestinal absorption insure GAKIC was assimilated du ⁇ ng the 60 on board over the 60 mmute pe ⁇ od of administration and testing (from -45 minutes through the end of the "15 mm" exercise set). For this reason, subjects were administered three equal doses over a 45 mmute preceding fatigue testing. This was to maximize the chances of having high muscle levels of GAKIC during the training bouts.
  • Evidence of supplemental effect during the "0 mmute" set narrows the effect time va ⁇ able to between 30 seconds and 46 minutes.
  • subjects are asked to produce submaximal efforts of quad ⁇ ceps force production by ⁇ ght knee extension at 90°/sec du ⁇ ng both the concent ⁇ c and eccent ⁇ c phases, with each phase again separated by a 15 second rest pe ⁇ od.
  • subjects are asked to produce maximal efforts of ⁇ ght knee extension at 90°/sec during both the concentric and eccent ⁇ c phases, with each phase again separated by a 15 second rest pe ⁇ od.
  • Subjects are given verbal encouragement throughout each maximal effort. Efforts continue until the same maximum peak torque curve has been achieved 3 times for each of the concentric as well as eccentric phase of muscle function.
  • Subjects are asked to return in one week for the second test visit. For this session, subjects are instructed to refrain from exercise of the lower extremity du ⁇ ng the entire three week testing pe ⁇ od. Subjects are asked to take a dietary record 24 hours preceding visit 2, and to fast for a period of 12 hours preceding subsequent visits.
  • Visit 2 Subjects have been instructed to fast (no food or beverages other than ad libitum water for 12 hours) p ⁇ or to the exercise session. At the beginning of this visit, subjects complete a 24 hour history, and dietary recall. After being seated for 15 minutes, resting heart rate and blood pressure are measured. Subjects first engage m a practice/warm-up session. Subjects are restrained with a lap belt and two shoulder harnesses and are asked to perform all repetitions with arms crossed over the chest. The practice session is comp ⁇ sed of 3 consecutive concent ⁇ c and eccent ⁇ c repetitions, followed by a second set of 3 maximal concent ⁇ c and eccentric repetitions. This practice session precedes delivery of GAKIC or placebo, to allow subjects to regain homeostasis prior to maximal testing.
  • a research assistant randomly assigns and mixes either 11.20 gm GAKIC (glycine-argmme- ⁇ -ketoisocaproic acid) or 9.46 gm sucrose isocalo ⁇ c placebo (pre- dispensed and coded by manufacturer) m 355 ml Marshx® low calorie Cranberry juice.
  • GAKIC glycine-argmme- ⁇ -ketoisocaproic acid
  • sucrose isocalo ⁇ c placebo pre- dispensed and coded by manufacturer
  • the 11.20 gm of GAKIC or 9.46 gm sucrose placebo is administered in three equal 3.73 gm doses (GAKIC) or three equal 3.15 gm doses (sucrose) at
  • Subjects document a w ⁇ tten record of dietary intake for subsequent analysis of consumed L- leucine, L-argmme, glycine, total protein, caffeine, total calories, and total carbohydrate, as for
  • Visit 2 Subjects are instructed to return in 14 days for visit 4.
  • Subjects document a w ⁇ tten record of dietary intake for subsequent analysis of consumed L-leucme, L-argmme, glyc e, total protein, caffeine, total calo ⁇ es, and total carbohydrate, as for Visit 2. Subjects are instructed to return in 14 days for Visit 5.
  • Visit 5 is identical to visit 3. Twenty-four hours following the treatment, fatigue testmg is performed as desc ⁇ bed for Visit 2, but is performed without delivery of GAKIC or placebo. Following 5 minutes of rest, the subject performs a set of 35 concentric and eccentric knee extension repetitions at 90°/sec. Data Collection/Analysis.
  • Measured torque is used as the dependent measure of force production. Torque produced du ⁇ ng the last 5 repetitions of each 35 repetition testing set is used to calculate a mean torque value for the last five repetitions. The mean of these 5 repetitions is divided by the Baseline Maximal concentric or Baseline Maximal eccentric torque values determined du ⁇ ng
  • the values collected and derived from the Km- com software are in units of joules produced during each set. Differences in the values of Fatigue Resistance ratio and total work between the GAKIC trials and the placebo trials are measured using repeated measures ANOVA or paired t-tests.
  • the withm-subjects group include treatment (GAKIC vs. placebo), and time following treatment oral administration (0, 5, 15 minutes, 24 hour). An a priori alpha significance level of 0.05 is used for all compa ⁇ sons.
  • Example 3 Total work performed during each fatigue set for the concentric phase
  • the TW value is a measure of a subject's ability to sustain total work output across time.
  • TW values (joules) were obtained by integrating the work performed during every repetition in a given set for both the concentric and eccentric phases of contraction. TW values could be affected by changes in the absolute force peak level, changes in ability to sustain a given force peak level, or changes in magnitude of sustained force output during each repetition.
  • GAKIC treatment resulted in greater concentric TW values compared to isocaloric carbohydrate treatment. At these times, GAKIC treatment increased the mean TW per set by 12%, 9%, and 11%, respectively, above isocaloric carbohydrate values.
  • the overall mean percent gain in muscle total work attributable to G.AKIC treatment was 10.5 ⁇ 0.8% retained for at least 15 min. See Figure 8.

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Abstract

The subject invention pertains to compositions and methods for enhancing muscle performance and recovery from fatigue in humans and animals. In a specific embodiment, the unique formulation is a glycine and L-arginine monohydrochloride salt of alpha-ketoisocaproic acid calcium (GAKIC).

Description

DESCRIPTION
USE OF KETOACIDS TOGETHER WITH AMINO ACIDS FOR ENHANCING MUSCLE PERFORMANCE AND RECOVERY FROM FATIGUE
Cross-Reference to a Related Application This application claims priority from provisional application USSN 60/065,429, filed November 13, 1997.
Background of the Invention
In the arena of athlete muscle performance, it is desirable to create conditions that permit competition or training at higher levels of resistance for a prolonged period of time. However, acute, intense anaerobic use of skeletal muscle often results in impaired athletic performance, with attendant losses in force and work output, and increased onset of muscle fatigue, soreness, and dysfunction. A single exhaustive exercise session—indeed, any acute trauma to the body such as muscle injury, resistance or exhaustive muscle exercise, or elective surgery— is characterized by perturbed metabolism that affects muscle performance in both acute and long term phases.
Exhaustive exercise depletes metabolic energy carbon sources and acutely disrupts skeletal muscle nitrogen metabolism in three principle ways. (1) Certain amino acids, including branched-chain amino acids, are released from muscle and are deaminated to elevate serum ammonia, oxidized locally as muscle fuel sources, and augment metabolic acidosis. (2) There is a decline in catalytic efficiency of muscle contraction events, as well as an alteration of enzymatic activities of nitrogen and energy metabolism. (3) Protein catabolism is initiated (including decreasing the rate of protein synthesis, as well as increasing degradation of non- contractible protein), thereby reducing long-term strength gains.
Recovery from fatigue during acute and extended exercise is associated with reversal of metabolic and non-metabolic fatiguing factors. Known factors that participate in human muscle fatigue, such as lactate, ammonia, and hydrogen ion, provide an incomplete and unsatisfactory explanation of the fatigue/recovery process, and it is likely that additional unknown agents participate (Baker, AJ., K.G. Koston, R.G. Miller, M.W. Weiner (1993) "Slow force recovery after long-duration exercise: metabolic and activation factors in muscle fatigue" J. Appl. Physiol. 74:2294-2300; Bazzarre, T.L., S.D. Murdoch, S.L. Uw, D.G. Herr, I.P. Snider (1992) "Plasma amino acid responses of trained athletes to two successive exhaustive trials with and without interim carbohydrate feeding" J. Am. Coll. Nutr. 11:505-511; Doh , G., GJ. Kasperek, E.B. Tapscott, H.A. Barkakat (1985) "Protein metabolism duπng endurance exercise" Fed Proc. 44:348-352; Edwards, R.H.T. (1983) "Biochemical basis of fatigue in exercise performance. Catastrophy theory of muscle fatigue" In: Biochemistry of Exercise, Proceedings of the Fifth International Symposium on the Biochemistry of Exercise (H.G. Kutrgen, J.A. Nogel, and J. Poormans, eds.); MacDougall, J.D., M.A. Tamopolsky, A. Chesley, S.A. Atkinson (1992)
"Changes in muscle protein synthesis following heavy resistance exercise m humans: a pilot study" Ada Physiol Scand. 146:403-404; and Walser, M., Ν.D. LaFrance, L. Ward, M.A. Nan Duyn (1987) "Progression of chronic renal failure in patients given ketoacids following ammo acids. Kidney Int. 32:123-128). α-ketoisocaproate (α-KIC) is the ketoacid parent chain of Z-leucme (i.e., it is Zleucine without the amino nitrogen), and is the first metabolite in the muscle catabolic pathway of leucme following reversible transammation to glutamate (Legaspi, A. (1989) "Adjunctive therapy for nutritional support in hospitalized patients" Nutr Clin. Pract 4:95-100). The intermediary metabolism of α-KIC and leucme plays a major role in regulatory muscle biochemistry, integrity, and physiology (Abrumrad, Ν.Ν., Wise, K.L., Williams, P.E. (1982)
"Disposal of α-ketoisocaproate: roles of liver, gut and kidneys" Am J. Physiol. 243:E123-E131; Buse, M.G. and S.S. Reid (1975) "Leucme: a possible regulator of protein turnover in muscle" J. Clin. Invest. 56:1250-1261; Flakoll, P.J., M.J. VanderHaar, G. Kuhlman, S. Nissen (1991) "Influence of α-ketoisocaproate on lamb broth, feed conversion, and carcass composition" J. Anim. Sci. 69: 1461-1467; Harper, A.E, R.H. Miller, K.P. Block (1984) "Branched-cham ammo acid metabolism" Ann. Rev. Nutr. 4:409-454; Jeevanandam, M., M.R. Ah, N.J Holaday, J.K. Weis, R. Peterson (1993) "Relative nutritional efficacy of argmme and omithine salts of α- ketoisocaproic acid in traumatized rats" Am J. Clin Nutr. 57:889-896; and Tamopolsky, M.A., S.A. Atkinson, J.D. MacDougall, B.B. Senor, P.W.R. Lemon, H. Schwartz (1991) "Whole body leucme metabolism duπng and after resistance exercises in fed humans" Med Sci Sports Exerc.
23:324-333).
In some instances erosion of muscle mass induced by overtraining or injury can be reduced by metabolic intervention (Blomstrand, E. and E.A. Newsholme (1992) "Effect of branched-cham ammo acid supplementation of the exercise-mduced change in aromatic ammo acid concentration in human muscle" Ada Physiol. Scand.46:293-298; and Moπ, E., M. Hasebe,
K. Kobayashi, H. Suzuki (1989) "Intermediate stimulation of protein metabolism m burned rats by total parenteral nutrition enriched m branched-cham ammo acids" J. Panenter. Enteral. Nutr. 13:484-489) with metabolites of leucme, although in humans leucme itself elevates serum and intramuscular ammonia as it is utilized as a local fuel (MacLean, D.A. and T.E. Grahm (1993) "Branched-cham ammo acid supplementation augments plasma ammonia responses duπng exercise in humans" J. Appl. Physiol. 74:2711-2717 and MacLean, D.A., T.E. Grahm, B. Saltin (1994) "Branched-chain amino acids augment ammonia metabolism while attenuating protein breakdown during exercise" Am. J. Physiol. 267:E1010-E1022). Ammonia (NH3) arises from the deamination of adenosine monophosphate to inosine monophosphate in the purine nucleotide cycle, as well as from the deamination of branched-chain amino acids. The ability of tissues to re-aminate leucine from supplemental α-ketoisocaproate has been clinically exploited as a means to treat muscle wasting in acutely traumatized and critically ill patients, while reducing their nitrogen load (Harper, A.E., R.H. Miller, K.P. Block (1984) "Branched-chain amino acid metabolism" Ann. Rev. Nutr. 4:409-454. Traumatized, critically ill hospital patients with eroding muscle mass and nitrogen wasting have been aided with adjuvant dietary intervention using analogs of branched-chain ketoacids and dibasic acids (Mitch, W.E. M. Walser, D.G. Sapir (1981) "Nitrogen sparing induced by leucine compared with that induced by its keto analogue, α-ketoisocaproate, in fasting obese men" J. Clin. Invest. 67:553-562; Sapir, D.G., M. Walser, E.D. Moyer (1983) "Effects of α-ketoisocaproate and of leucine on nitrogen metabolism in postoperative patients" Lancet 7: 1010-1014; and Warren, BJ., M.H. Stone, JJ. Kearney, SJ.
Fleck, R.L. Johnson, G.D. Wilson, W.J. Kraemer (1992) "Performance measures, blood lactate and plasma ammonia as indicators of overwork in elite junior weight lifters" Int. J. Sports Med. 13:372-376). Hospital and in vitro studies with medical patients exhibiting liver disease and attendant central portal encephalopathy, or renal disease, show that certain administered combinations of ketoacid/amino acid complexes improve muscle trauma recovery time, reduce serum ammonia, enhance injury repair, and yield long-term catabolic/anti-anabolic effects on muscle protein. Acute changes in biceps brachii or quadriceps femoris muscle inter-conversion of KIC and leucme occurs following heavy resistance training (MacDougall et al.. (1992) "Changes in muscle protein synthesis following heavy resistance exercise in humans: a pilot study" Ada Physiol. Scand. 146:403-404).
Enhancing muscle recovery following trauma occurs not simply by administering oral or intravenous leucine alone, but instead it responds to increasing the steady-state concentration of α-ketoisocaproic acid. This anabolic ketoacid is a major factor in reducing protein catabolism, stimulating muscle synthesis, and sparing glucose oxidation, while stimulating insulin release. Indeed, α-ketoisocaproic acid is superior to leucine in this regard in human and rat muscle studies (Buckspan, R., B. Hoxworth, E. Cersosimo, J. Devlin, E. Horton, N. Abrumrad (1986) "Alpha-ketoisocaproate is superior to leucine in sparing glucose utilization in humans" Am. J. Physiol. 25 E648-E653).
The observed biochemical and physiological effects of oral leucine/α-KIC on muscle recovery involve several enzymes (Ahring, R.P., K.P. Block, M.G. Buse (1986) "Leucme and isoleucine activate skeletal muscle branched-chain α keto acid dehydrogenase in vivo" Am. J. Physiol. 250:E599-E604; Buse, M.G. and S.S. Reid (1975) "Leucine: a possible regulator of protein turnover in muscle" J. Clin. Invest. 56:1250-1261; and Kasperek, GJ. (1989) "Regulation of branched-chain 2-oxo acid dehydrogenase activity during exercise" Am. J. Physiol. 256:E186-E190.): The most critical enzymes are BCAA-aminotransferase, the BCKA dehydrogeneases family, L-leucine dehydrogenase, and 3-methyl-2-oxobutanoate dehydrogenase. Enzyme concentrations of BCAA-aminotransferase are relatively unregulated at fairly steady-state levels in muscle. Therefore, transamination is reversibly catalyzed by BCAA-aminotransferase activity through mass action of available concentrations of α- ketoisocaproate, L-leucine, L-glutamate, α-ketoglutarate, and their ancillary metabolites. In the presence of the excessive NH3 liberated from the purine nucleotide cycle activated during exercise or from glutamate dehydrogenase, L-leucine dehydrogenase provides a beneficial pathway that catalyzes the NH3 amination of α-ketoisocaproate to yield L-leucine. The enzyme 3-methyl-2-oxobutanoate dehydrogenase can catalyze the decarboxylation of α-ketoisocaproate, leading to pathways eventually creating acetoacetate. Alpha-ketoisocaproic acid can be hydrolyzed to beta-hydroxy-beta-methylbutyrate via α-ketoisocaproate dioxygenase. (Harper, A.E., R.H. Miller, K.P. Block (1984) "Branched-chain amino acid metabolism" Ann. Rev. Nutr. 4:409-454; Van-Koevering, M., S. Nissen (1992) "Oxidation of leucine and alpha- ketoisocaproate to beta-hydroxy beta-methylbutyrate in vivo" Am. J. Physiol. 262(1 pt 1):E27- 31). It has been suggested that beta-hydroxybeta-methybutyrate may be involved in partially preventing muscle degradation and promoting muscle gain in chronic resistance training (Nissen, S. et al. (1996) "Effect of leucine metabolite beta-hydroxy-beta-methylbutyrate on muscle metabolism during resistance-exercise training" J. Appl. Physiol. 81 :2095-2104). In contrast to unregulated BCAA-aminotransferase, the activity of BCKA-dehydrogenase is highly regulated during exercise. In muscle and liver, BCKA-dehydrogenase is a multienzyme complex that catalyzes the irreversible oxidative decarboxylation of BCKA, as it reduces NAD to NADH. The activity of BCKA-dehydrogenase greatly increases immediately after strenuous exercise, with subsequent return to resting baseline levels by 10 minutes post-exercise (Kasperek, GJ. (1989) "Regulation of branched-chain 2-oxo acid dehydrogenase activity during exercise" Am. J. Physiol. 256:E186-E190). BCKA-dehydrogenase enzyme activity is regulated by an ATP phosphorylation (inactivation)-dephosphorylation (activation) mechanism, α-ketoisocaproate is a key stimulator of this enzyme complex, whereby it inhibits the ATP -mediated kinase allosteric inactivation of BCKA-dehydrogenase. The potency of α-ketoisocaproate is several orders of magnitude greater than any other BCKA BCKA-dehydrogenase activity is therefore mediated by exercise and nutritional factors at the levels of allosteric and substrate mass action. Control of muscle dysfunction with intravenous or dietary amino acids administered in the purified "free" form, especially branched-chain amino acids (BCAA), has been attempted in many studies (R.S. Bailey (1993) "Commercially marketed supplements for bodybuilding athletes" Sports Med. 15:90-103; and MacLean, D.A., T.E. Grahm, B. Saltin (1994) "Branched- chain amino acids augment ammonia metabolism while attenuating protein breakdown during exercise" Am. J. Physiol. 267:E1010-E1022). There are no substantiated guidelines for orally supplemented branched-chain amino acids because there have been no reputable double-blind controlled studies conducted to establish their effects; the use of BCAA in athletes is largely anecdotal and without systematic testing of performance. In contrast, however, it has been clearly established that intramuscular protein catabolism is decreased, protein synthesis is increased, and serum ammonia is decreased by metabolic intervention with branched-chain α- ketoacid (BCKA) analogs of BCAA, and that their effect may be enhanced by simultaneous administration of amino acids (Pui, Y.M.L. and H. Fisher (1979) "Factorial supplementation with arginine and glycine on nitrogen retention and body weight gain in the traumatized rat" J. Nutr. 109:240-246; and Smith, K. and MJ. Rennie (1990) "Protein turnover and amino acid metabolism in human skeletal muscle. In: Baillier's Clinical Endocrinology and Metabolism 4:461-498).
L-Arginine is considered a "conditionally essential" amino acid that becomes essential under certain metabolic conditions including muscle trauma and injury (Dundrick, P.S. and W.W. Souba (1991) "Amino acids in surgical nutrition" Surg. Clin. N Am. 71:459-476 and
Smith, K. and MJ. Rennie (1990) "Protein turnover and amino acid metabolism in human skeletal muscle. In: Baillier's Clinical Endocrinology and Metabolism 4:461-498). L-Arginine is a dibasic amino acid that participates in the urea cycle and other intermediary pathways, notably serving as the starting substrate for biosynthesis of polyamines. Polyamines are essential for protein synthesis, and cell growth and proliferation. Arginine synergetically promotes the nitrogen-retaining effects of α-KIC. Studies show that cationic analogs such as ornithine, citrulline, or lysine may not be as effective as arginine, and may indeed inhibit the cell membrane transporters serving arginine (Kilberg, M.S., B.R. Stevens, D.A. Novak (1993) "Recent advances in mammalian amino acid transport" Annu. Rev. Nutr. 13:137-165; Mitch, W.E., M. Walser, D.G. Sapir (1981) "Nitrogen sparing induced by leucine compared with that induced by its keto analogue, α-ketoisocaproate, in fasting obese men" J. Clin. Invest. 67:553- 562; Pan, M., W.W. Souba, B.R. Stevens (1992) "Independent regulation of arginine and alanine transporters in human intestinal epithelial Caco-2 cells" The Physiologist 35(5):A-15; Pan, M., B.R. Stevens, W.W. Souba (1994) "Regulation of intestinal amino acid transport: A surgical perspective" Contemp. Surg. 44:213-220; Pan, M., M. Malandro, B.R. Stevens (1995) "Regulation of System y+ arginine transport capacity in differentiating human intestinal Caco-2 cells" Am. J. Physiol. 268:G578-G585; and Pan, M. and B.R. Stevens (1995) "Differentiation- and protein kinase C-dependent regulation of alanine transport via system B" J. Biol. Chem. 270:3582-3587). Previous studies showed that omithine, lysine, and histidine salts of α-ketoglutaric acid, beta-hydroxy-beta methylbutyric acid, α-keto-beta-methylvaleric acid, and other ketoacid analogs of branched-chain amino acids promote positive nitrogen balance (Nissen et al. (1996) "Effect of leucine metabolite beta-hydroxy-beta-methylbutyrate on muscle metabolism during resistance-exercise training" J. Appl. Physiol. 81 :2095-2104; Van-Koevering, M., S. Nissen (1992) supra; Cynober, L. (1991) "Omithine α-ketoglutarate in nutritional support" Nutrition
7(5):313-322; Cynober, L., M. Naubourdolle, A. Dore, J. Giboudequ (1984) "Kinetics and metabolic effects of orally administered omithine α-ketoglutatarate in healthy subjects fed with a standard regimen" Am. J. Clin. 39:514-519; Funk, M.A., K.R. Lowry, D.H. Baker (1987) "Utilization of the L- and DL-stereoisomers of a α-keto-β-methyl valeric acid by rats and comparative efficacy of the keto analogs of ranched-chain amino acids provided as omithine, lysine, and histidine salts" J. Nutr. 117:1550-1555; Sitren, H.S. and H. Fisher (1977) "Nitrogen retention in rats fed on diets enriched with arginine and glycine" Br. J. Nutr. 37: 195-208; Visek, W.J. (1986) "Arginine needs, physiological state and unusual diets: A reevaluation" /. Nutr. 116:36-46; and Wolf, J.G. Staleness. In Encyclopedia of Sports Sciences and Medicine, L.A. Larson and D.E. Hermann (Eds) New York: MacMillan Publishing Co. (1971) pgs. 1048-1050).
Leucine or other BCAA alone did not demonstrate this effect (Buckspan, R., B. Hoxworth, E. Cersosimo, J. Devlin, E. Horton, N. Abrumrad (1986) "Alpha-ketoisocaproate is superior to leucine in sparing glucose utilization in humans" Am. J. Physiol. 251:E648-E653; Cersosimo, E., B.M. Miller, W. Lacy, N.N. Abrumrad (1983) "Alpha-ketoisocaproate, not leucine, is responsible for nitrogen sparing during progressive fasting in normal male volunteers" Surg.
Forum 43:96-98; Sandstedt, S., L. Jorfeldt, J. Larsson (1992) "Randomized, controlled study evaluating effects of branched chain amino acids and α-ketoisocaproate on protein metabolism after surgery" Br. J. Surg. 79:217-220). Therapeutic strategies for recovery of medical or longitudinal muscle trauma have recently demonstrated the synergistic importance of dibasic amino acid salt complexed with ketoacids. Traumatized, critically ill hospital patients with chronic eroding muscle mass and nitrogen wasting have been aided with adjuvant dietary intervention using analogs of branched-chain ketoacids and dibasic acids (M tch, W.E., M. Walser, D.G. Sapir (1981) "Nitrogen sparing induced by leucine compared with that induced by its keto analogue, α-ketoisocaproate, in fasting obese men" J. Clin. Invest. 67:553-562; Sapir, D.G., M. Walser, E.D. Moyer et al. (1983) "Effects of α-ketoisocaproate and of leucine on nitrogen metabolism in postoperative patients" Lancet 7:1010-1014; Warren, BJ., M.H. Stone, J.T. Kearney, SJ. Fleck, R.L. Johnson, G.D. Wilson, W.J. Kraemer (1992) "Performance measures, blood lactate and plasma ammonia as indicators of overwork in elite junior weight lifters" Int. J. Sports Med. 13:372-376).
Brief Summary of the Invention The subject invention concerns methods and compositions for enhancing muscle performance and recovery from fatigue. More specifically, the subject invention concerns a unique metabolic intervention to improve athletic dynamic muscle strength and muscle work, and recovery from acute exhaustive anaerobic strength training exercise.
In a preferred embodiment the subject invention concerns the use of a glycine and L- arginine monohydrochloride salt of alpha-ketoisocaproic acid calcium (GAKIC) via oral treatment to a human or animal to enhance muscle performance and recovery from fatigue.
GAKIC has the following advantages:
1) GAKIC treatment increases the ability to sustain athletic muscle force during intense anaerobic muscle exercise;
2) GAKIC treatment increases the ability to sustain athletic muscle work during intense anaerobic muscle exercise; and 3) GAKIC increases the overall muscle performance by decreasing muscle absolute fatigue while retarding the rate of muscle fatigue.
In accordance with the subject invention, oral metabolic intervention with GAKIC significantly improves athletic muscle performance compared to control isocaloric carbohydrate: (1) G.AKIC treatment increases the ability to sustain muscle force (torque) up to at least 28% during intense acute anaerobic dynamic muscle exercise; (2) GAKIC treatment increases the ability to sustain muscle total work (joules) by up to 12% during the early phase of intense anaerobic dynamic muscle exercise; and (3) GAKIC increases the overall muscle performance by forestalling muscle fatigue during the early phases of anaerobic exercise.
The subject invention also concerns a new testing protocol which is able to objectively test, quantify and provide a reproducible assessment of muscle work, fatigue, and recovery of dynamic muscle function associated with acute anaerobic exercise. This protocol was exploited to discriminate the effects of metabolic interventions on muscle function during training regimes.
The compositions and methods of the subject invention can be used to treat humans and animals. In one embodiment the materials and methods are used to treat horses. Brief Description of the Drawings Figure 1 shows a brief overview of the whole-body intermediary metabolic relationship among BCAA and BCKA.
Figure 2 shows the testing protocol overview. Figure 3 shows a representative computation of the fatigue resistance index (FRI).
Figure 4 shows the concentric muscle fatigue resistance enhancement by GAKIC. Figure 5 shows the percent gain in fatigue resistance directly attributable to GAKIC treatment.
Figure 6 shows the total work performed during each fatigue set for the concentric phase.
Figure 7 shows the work gained from GAKIC treatment.
Figure 8 shows the percent increase in total work gained from GAKIC treatment.
Detailed Disclosure of the Invention The subject invention provides materials and methods that enhance muscle performance under conditions of acute exhaustive anaerobic dynamic exercise. The subject invention can be used to enhance human or animal muscle performance. Animals which can benefit from the subject invention include horses.
GAKIC, a novel formulation useful according to the subject invention, is safe for humans and animals. In a preferred embodiment, GAKIC comprises glycine, L-arginine, and α-ketoisocaproic acid. GAKIC can be used according to the subject invention as a nutritional supplement to enhance athletic performance. The benefits of GAKIC include at least the following:
1) A more rapid and complete recovery from intense acute athletic training, greater acute muscle power, and ultimately improved performance is achieved with GAKIC.
2) GAKIC is a safe means to enhance athletic performance, without the harmful side effects or illegality of anabolic steroids, pharmaceuticals or elevated caffeine. 3) In highly trained competing athletes, the significant muscle performance gain directly attributable to GAKIC can provide a winning edge among nearly equal competitors. 4) GAKIC can be useful to medical patients with sports injuries, as a means to improve exercise tolerance and recovery during rehabilitation. 5) The described exercise and testing protocol using the computerized isokinetic dynamometer is a unique and useful means to quantify/assess muscle force, work, and dynamic fatigue performance during concentric and eccentric contractions under well-controlled reproducible experimental conditions. These conditions are directly extrapolated to the real-world of athletic training and competition.
6) The described exercise and testing protocol using the computerized isokinetic dynamometer is a unique and useful means to quantify/assess muscle function in medical patients with sports injuries, as a means to improve exercise tolerance and recovery during rehabilitation.
7) The benefits of GAKIC can help improve animal jumping and racing performance.
In addition to, or instead of, Z-arginine, the composition of the subject invention can comprises other cationic or dibasic amino acids. These amino acids may be, for example, omithine, histidine, and/or lysine. In a further embodiment of the subject invention, glutamate and/or glutamine may be substituted for glycine. Also, other salts of alpha-ketoisocaproic acid may be used in the compositions of the subject invention. These salts may be, for example, the sodium or potassium salts. Alternatively, the free acid may be used. It should be noted that the subject invention is not limited only to the use of alpha-ketoisocaproic acid and its salts. Other related acids and salts may also be used. These other related acids and salts may be, for example, alpha-keto-isovaleric acid, alpha-keto-beta-methylvaleric acid, or pyruvic acid as the free acids or as a salt of calcium, potassium sodium, or as a conjugate.
The components of the compositions of the subject invention may be administered individually, as a mixture, or as covalent bonds, or other conjugate. For example, alpha-keto- isocaproic acid or alpha-keto-isovaleric acid, alpha-keto-beta-methylvaleric acid, or pyruvic acid can be conjugated with arginine. Thus, in a preferred embodiment, the subject invention pertains to the use of a ketoacid with a cationic amino acid to improve muscle performance and recovery from fatigue. Although L-isomers are preferred racemic mixtures may also be utilized.
One aspect of the subject invention pertains to a stable tripeptide comprised of glycine, L-arginine, and α-ketoisocaproate covalently linked by amide bonds. Advantageously, this orally consumed GAKIC precursor is readily hydrolyzed by human pancreatic proteases, yielding the appropriate components of GAKIC in vivo in the intestinal lumen or in vitro.
In one embodiment, a low calorie beverage is provided. In a specific embodiment, low calorie cranberry juice can be used. In one embodiment, the components can be orally administered via capsules. Oral administration of the subject glycine-arginine-α-ketoisocaproic acid formulation is preferred over the intravenous route, due to the coordinated metabolic communication (Figure 1) between skeletal muscle, liver, kidneys and the splanchnic bed, notably the small intestine. Both the splanchnic and skeletal muscle regions are the body's major sites of branched-chain amino acid metabolism. Although oral administration is considered the best route, the intravenous route is available in subjects unable to receive oral administration.
Clinical studies show that human subjects can tolerate a 30 gm oral single bolus of single dibasic amino acids (e.g., L-arginine monohydrochloride), branched-chain amino acids, or 35 mmole/day of alpha-ketoacid (Barbul, A. (1985) "Arginne: Biochemistry, physiology, and therapeutic implications" JPEN 10:227-238 and Ceira, F.B. (1992) "Role of nutrition in the management of malnutrition and immune dysfunction of trauma" J. Am. Coll. Nutr. 11:512-518). Arginine has been administered intravenously at 30 gm per day for over a week with no adverse effects (Barbul, A. (1985) supra). Ketoacids have been given at over 10 gm per day for up to 10 months (Walser, M., N.D. LaFrance, L. Ward, M.A. Van Duyn (1987) "Progression of chronic renal failure in patients given ketoacids following amino acids. Kidney Int. 32: 123-128).
Safety guidelines (Cerra, F.B. (1992) supra) recommended nutrient loading to less than or equal to 1.5-2.0 gm amino acid per kg body weight per day, that is, 105 gm - 140 gm per day for a typical 70 kg human. Doses beyond this amount may result in a possible benign side effect, namely gastrointestinal discomfort. The stated doses disclosed herein are much less than the stated upper limit safety range for a typical 70 kg subject (Benevenga, NJ. and R.D. Steele
(1984) "Adverse effects of excessive consumption of amino aciάs" Ann. Rev. Nutr. 4:157-181 and Sandstedt, S., L. Jorfeldt, J. Larsson (1992) "Randomized, controlled study evaluating effects of branched chain amino acids and α-ketoisocaproate on protein metabolism after surgery" Br. J. Surg. 79:217-220), and are not given as a single bolus, but instead are distributed as fractional aliquots over a 45 to 75 minute period, and therefore are unlikely to cause gastrointestinal discomfort. In one embodiment the dosage may be as large as tolerated per the recommended safety guidelines.
The components of the compositions of the subject invention can be administered in an effective dosage which can be readily ascertained by a person skilled in the art having the benefit of the current disclosure. A person skilled in the art would appreciate that the ratios as well as the concentrations of components can be modified in order to achieve the desired effect of enhanced muscle performance and reduced muscle fatigue.
One aspect of the subject invention concerns unique protocols and assessment instruments which can be used to quantify the effects of metabolic intervention on muscle force, work, and fatigue associated with acute anaerobic exercise. These measurements standardize the results from each individual subject, thereby permitting meaningful compaπsons withm the repeated-measures randomized crossover design. In the literature, isokinetic fatigue is commonly assessed by measuπng the number of repetitions a subject can perform before force levels drop below a certain standard percentage of peak torque, comparison of complete exhaustion relative to a maximal static force, or assessment of time to exhaustion (Lewis, S.F.,
CS Fuko (1998) "A new approach to studying muscle fatigue and factors affecting performance duπng dynamic exercise in humans" Exercise Sports Med Rev 26:91-116) In one aspect, the current invention provides a novel Fatigue Resistance Index (FRI) which provides a uniform measure of dynamic fatigue resistance with respect to each subject's individual force output. The FRI provides a meaningful measure of the dynamic resistance to fatigue by allowing all subjects to perform the same concentric and eccentπc fatigue protocol of four 35 -repetition high intensity sets of 90°/sec knee extensions, regardless of fatigue rate. The protocol and assessment are in contrast to other methods which have attempted using isokinetic measurements to quantify exercise fatigue or by using bicycle ergometers, free weight trials, vertical leap tests, and treadmill running; these methods are not as sensitive to acute dynamic changes in muscle force.
Pre-fatigue and post-fatigue peak torque and total work values were measured in isolated quadπceps femoπs of subjects using a computer-controlled isokinetic dynamometer over a 23 day interval. Duπng testing, fasted subjects were given oral GAKIC or isocaloπc carbohydrate over a 45 mm period preceding exercise. Subjects then immediately performed four 35- repetition high intensity sets of 90°/sec knee extensions on the isokinetic dynamometer employing concentric and eccentπc contractions. Measurements were made before and during the fatigue protocol. The effects of the treatments were tested in a randomized double-blmd crossover repeated measures design (N=13 subjects). Additional parameters were maintained constant throughout the protocol: systolic and diastolic blood pressure, body weight, legg mass, and dietary L-leucme, L-arginme, glycine, total protein, total calories, total carbohydrate and caffeine. A novel Fatigue Resistance Index (FRI=[peπ-exhaustιon torque]/[baselme peak torque]) and a total work (TW) assessment (total work produced for the duration of each 35- repetition set) were each obtained duπng the muscle's concentric and eccentric phases for each set. This provided changes m fatigue over time. The mean baseline concentric isokinetic knee extension maximal torque was 240 ± 9 Nm. The FRI and TW for each of the 35-repetιtιon sets obtained at 0, 5, and 15 mm following oral GAKIC was greater than for isocaloπc carbohydrate (control) At these times, GAKIC treatment increased the mean FRI by 28%, 21%, and 13% above isocaloπc carbohydrate values, respectively, while the TW per set was increased by GAKIC by 12%, 9%, and 11% respectively, above isocaloπc carbohydrate values (overall mean percent gam m muscle TW attributable to GAKIC was 10.5 ± 0.8% retained for at least 15 mm.). After 24 hr, both GAKIC and placebo concentric forces returned to the same absolute values with mean FRI = 0.41 ± 0.05 (98 N-m/240 N-m), and mean TW = 4600 ± 280 joules. Analysis of values by order of treatment in the repeated measures crossover design (i.e., GAKIC or carbohydrate given in either first or second set of trials) indicated that there were no significant differences (P > 0.05) attributable to the random order of testing. Compared to lsocaloπc carbohydrate, GAKIC treatment increased the muscle force and work generated during acute anaerobic exercise, while retarding the rate of fatigue, as assessed using the stated protocol.
The subject invention is also useful m extended, continual aerobic phases of intense exercise exceeding 15 mm, but less than 24 hr of continuous exercise, that include both anaerobic glycolytic as well as oxidative metabolism due to the metabolic pathways employing the components of GAKIC. Thus, GAKIC can also be used for longitudinal weight-trammg.
Materials and Methods Basic design. The effects of GAKIC were studied using a repeated measures, randomized, crossover scheme conducted a double-blind fashion, with subjects receiving both oral GAKIC (treatment) and oral lsocaloπc carbohydrate (sucrose placebo control) solutions, and with treatment measurements assessed against a non-treatment baseline established for each subject. Significant differences and trends were assessed by repeated measures ANONA. and/or paired t-tests. Data were recorded in real-time using Kin-Corn 125AP isokinetic dynamometer software (Chattanooga Group, Inc., Chattanooga, TΝ)
Human subjects were recruited evenly between males and females from among all races and ethnic groups. The final pool of subjects (Ν=13) were all healthy male athletes involved in resistance training for a minimum of 6 months preceding the study. Subjects were free from the following metabolic conditions: diabetes-melhtus, aminoaciuπas including maple syrup urme disease (congenital defect in branched-cahin ketoacid dehydrogenase), renal failure, muscle wasting, hypertension, abdominal radiotherapy or intestinal resection, fever, steroid or lmmunosuppressant use. Duπng the testing period subjects were monitored for dietary intake of L-leucme, L-arginine, glycine, total protein, total calories, total carbohydrate and caffeine. No significant differences (p > 0.05) were found between the GAKIC and carbohydrate treatment peπods for any dietary vaπable. Blood pressure, leg weight, and body weights were also monitored and determined to be constant (P > 0.05) duπng the testing peπod; subjects were randomly assigned to each group, and were remunerated after completion of the entire series of visits.
Subject physical characteπstics were measured during the randomly assigned carbohydrate and GAKIC treatment peπods Because the crossover design used the exact same subjects for both treatments, subject age (21 ± 2) and height (72 ± 2 inches) were the same. The carbohydrate and GAKIC treatment periods were not significantly different (p > 0.05) with respect to body weight (82.93 vs. 82.74 lbs), limb weight (84.77 vs 86.15 Newtons), or m blood pressure immediately preceding to testing (117/74 vs. 119/73 mm Hg; systohc/diastolic). Treatments. Each 11.20 gm of glycme-L-argmine-α-ketoisocaproic acid ("GAKIC") treatment formulation compπsed 2.0 gm (17.85 wt %) of glycine, 6.0 gm (53.57 wt %) of L- argmme monohydrochloride, and 3.2 gm (28.57 wt %) of α-ketoisocaproic acid di-calcium. Subjects were given a solution to dπnk containing 11.20 gm freshly dissolved powdered GAKIC or 9.46 gm sucrose isocaloπc placebo in 355 ml Pubhx®Reduced caloπe Cranberry Juice (62% less calories than regular cranberry juice). This effectively provided the same taste m control and treatment solutions, as determined by hedonic assessment. Solutions were consumed in 3 ahquots over 45 minutes.
Protocol. Each subject was required to make five visits to the testing facility, with three visits in the fasted state. Force measurements and total work measurements were recorded with a Kin-Com 125AP isokinetic dynamometer. This computeπzed device permitted continual assessment of each subject's ability to produce dynamic muscular force and work over time. Peak torque, average torque, and work were analyzed from a full set of repetitions throughout every degree in the range of motion. An overall concept diagram of the protocol is shown in Figure 2. The Fatigue Resistance Index. The Fatigue Resistance Index (FRI) descπbes the maximal exerted force sustained under fatiguing conditions as a percent of maximal force exerted under fresh conditions for each individual over a period of time. FRI is based on the ability to produce concentπc or eccentπc torque immediately following maximal exertion over 30 initial repetitions of a 35 repetition set in the presence of metabolic treatment, compared to that same individual's ability to produce force under fresh (baseline) conditions in the absence of treatment. The fixed value of 35 maximal isokinetic concentπc/eccentπc repetitions to induce fatigue in the quadriceps muscle groups was determined based on a pilot study. A pilot study was initially employed to determine isokinetic fatigue conditions and muscle performance quantifications. Based on this pilot, fatigue was induced m the quadπceps muscle group duπng a fixed value of 35 maximal isokinetic concentric/eccentric repetitions. One week preceding initial fatigue testing, a Baseline Isokinetic Knee Extension Maximal Strength Value ("BIKEMSV", m peak torque units of Nm) was obtained at a rate of 90°/sec both concentrically and eccentπcally. Duπng subsequent fatigue test bouts associated with the metabolic treatments, the subjects then performed another sequence of 35 continuous isokinetic concentric/eccentric knee extension repetitions. Peak forces for both the concentric and eccentric phases obtained duπng the last 5 repetitions of each 35 rep set (i.e., pen-exhaustion torque) were analyzed. An average of these five peak values was calculated for both the concentric and eccentric phase of the muscular event. The FRI was calculated by dividing this average value by the maximal force generated duπng the BIKEMSV testing. Concentric and eccentric BIKEMSV values were determined for each subject duπng "Visit 1," described below. FRI was obtained for each set in the protocol (0', 5', 15', and 24 hr).
Total Work. Total work (TW) values for each 35 repetition set were also collected and deπved from the Km-Con 125AP software, and are expressed m units of joules produced duπng each set. Isokinetic Dynamometer Instrumentation. Each subject's right knee was carefully aligned with the rotation axis of the dynamometer lever arm, and subjects were secured in place using a waist strap, two should straps, and a thigh stabilizer. The distal shm pad was adjusted for each subject and placed two finger breadths above the lateral malleolus. The shm pad was securely fastened to the level arm using a Velcro strap. Gravity compensation procedures were earned out according to manufactures recommendations with the knee m and extended position.
Knee extension and flexion motion occurred in a 75 ° arc, so that start and stop angles were 85 ° and 10°, respectively.
Differences m the values of FRI and TW between the GAKIC trials and the lsocaloπc carbohydrate control trials were measured using repeated measures ANOVA or paired t-tests. Also assessed was the effect of treatment order. An a pπoπ alpha significance level of 0.05 was used for all compaπsons.
A Fatigue Resistance Index (FRI=[peπ-exhaustιon torque j7[baselme peak torque]) and a total work (TW) assessment (total work produced for the duration of each 35-repetιtιon set) was obtained duπng the muscle's concentric and eccentric phases for each set. This provided changes in fatigue over time. The FRI and TW for each of the 35-repetιtιon sets obtained at 0,
5, and 15 mm following oral GAKIC was greater (PO.02) than for sucrose (control). At these times, GAKIC treatment increased the mean FRI by 28%, 21%, and 13% above carbohydrate values, while the TW per set was increased by GAKIC by 12%, 9%, and 11% above carbohydrate values. After 24 hr, both GAKIC and carbohydrate concentric forces returned to the same absolute values (P>0.05) with mean FRI=41± 5% (98 N»m/240 N-m), and mean
TW=4600 ± 280 joules. Analysis of values by order of treatment in the repeated measures crossover design (i.e., GAKIC or carbohydrate given in either first or second set of trials) indicated that there were no significant differences (P>0.05) attributable to the random order of testing. Statistical Considerations
Basic Design. This study is a repeated measures, randomized, crossover design conducted m a double-blmd fashion, with subjects receiving both oral GAKIC (treatment) and oral sucrose (control placebo) solutions, and with treatment measurements assessed against a non-treatment baseline established for each subject. Significant differences and trends are assessed by repeated measures ANOVA and/or paired t-test using SigmaStat™ analysis software
Randomization of "double-blmd" procedure. Subjects were randomly assigned to each group by computer-generated randomization. An independent research associate kept records confidential and "blind" from the investigators until after data were analyzed.
Sample size. The number of subjects recruited conservatively exceeds the calculated sample size requirement from Power Analysis using the following conditions. These parameters were deπved from preliminary expeπments using a leg extension force apparatus and protocol: the detectable percent difference m means between two groups is better than a Force Resistance Index = 5, with a population standard deviation of ± 5. With the power set at 0.80 and with α=
0.05, then N=10 for a repeated measures paired t-test. The actual final sample size giving the present data was N=13.
The Expenmental Model: Testing effects. When implementing a testing protocol on a naive subject, especially one utilizing an uncommon movement type like isokinetic exercise, there is always concern that the results might be affected by a testing or learning effect. This is an improvement m performance that may occur as a result of experience and coordination gained through multiple trials. The double-blind, repeated measures crossover design of this study was invoked to negate such possible testing effects. This design gave each group equal chance to be affected, therefore canceling out any impact of significance; there were no significant (p > 0.05) differences m any treatment attributable to the random order of treatment
(G.AKIC or carbohydrate). In addition, the blinded testers administered a two set warm up teaching/practice phase to subjects each testing day preceding fatigue tπals. Subjects performed 3 consecutive submaximal isokinetic πght knee extensions at 90°/sec, followed by a maximal set of 3 repetitions. Snow and Blackhn (Snow, C. J., K. Blackhn (1992) "Reliability of knee flexor peak torque measurements from a standaπzed test protocol on a Km/Com dynamometer"
Arch. Phys. Med. Rehabil. 73:15-21) found much higher test-retest correlations when including within-session maximal contractions in warm-up procedures, as compared to between session practice. Thus, these small bouts served more as a coordination warm up than a physical one. These techniques were successful protecting against a testing effect. When grouped by chronological order rather than according to treatment, there were no between-group differences for FRI nor TW (P > 0.05).
The Expenmental Model: Time variable. The speed at which the GAKIC supplement is absorbed and distπbuted to the skeletal muscle is not completely known, but typical rates of gastπc emptying and intestinal absorption insure GAKIC was assimilated duπng the 60 on board over the 60 mmute peπod of administration and testing (from -45 minutes through the end of the "15 mm" exercise set). For this reason, subjects were administered three equal doses over a 45 mmute preceding fatigue testing. This was to maximize the chances of having high muscle levels of GAKIC during the training bouts. Evidence of supplemental effect during the "0 mmute" set narrows the effect time vaπable to between 30 seconds and 46 minutes.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Example 1 — Testing Procedures Visit 1.
During the initial visit, subjects complete a medical history questionnaire and are screened by project personnel to ensure concurrence with the inclusion/exclusion cπteπa. A maximal force-repetition baseline is initially obtained three times for each subject to measure changes in maximal force production and to quantify fatigue over time m ensuing experiments. Subsequent fatigue testing utilizes this value for both control and GAKIC treatments. The same-subject random crossover design of this project eliminates any minor biases that pretesting could potentially introduce. Subjects are positioned on the Km-Com 125AP isokinetic dynomometer. Once positioned, subjects are asked to produce submaximal efforts of quadπceps force production by πght knee extension at 90°/sec duπng both the concentπc and eccentπc phases, with each phase again separated by a 15 second rest peπod. Following this warm-up exercise, subjects are asked to produce maximal efforts of πght knee extension at 90°/sec during both the concentric and eccentπc phases, with each phase again separated by a 15 second rest peπod. Subjects are given verbal encouragement throughout each maximal effort. Efforts continue until the same maximum peak torque curve has been achieved 3 times for each of the concentric as well as eccentric phase of muscle function.
Subjects are asked to return in one week for the second test visit. For this session, subjects are instructed to refrain from exercise of the lower extremity duπng the entire three week testing peπod. Subjects are asked to take a dietary record 24 hours preceding visit 2, and to fast for a period of 12 hours preceding subsequent visits.
Visit 2 Subjects have been instructed to fast (no food or beverages other than ad libitum water for 12 hours) pπor to the exercise session. At the beginning of this visit, subjects complete a 24 hour history, and dietary recall. After being seated for 15 minutes, resting heart rate and blood pressure are measured. Subjects first engage m a practice/warm-up session. Subjects are restrained with a lap belt and two shoulder harnesses and are asked to perform all repetitions with arms crossed over the chest. The practice session is compπsed of 3 consecutive concentπc and eccentπc repetitions, followed by a second set of 3 maximal concentπc and eccentric repetitions. This practice session precedes delivery of GAKIC or placebo, to allow subjects to regain homeostasis prior to maximal testing.
Upon arπval of the subject, a research assistant randomly assigns and mixes either 11.20 gm GAKIC (glycine-argmme-α-ketoisocaproic acid) or 9.46 gm sucrose isocaloπc placebo (pre- dispensed and coded by manufacturer) m 355 ml Publix® low calorie Cranberry juice. This is a double-blmd study, and thus the examiners are unaware of which supplement is being administered only the research assistant records this information for use by the investigators following statistical analysis. The 11.20 gm of GAKIC or 9.46 gm sucrose placebo is administered in three equal 3.73 gm doses (GAKIC) or three equal 3.15 gm doses (sucrose) at
-45 (minus 45) minutes, -20 (minus 20) minutes, and 0 minutes prior to Quadπceps Fatigue Testing on the Kin-Com 125AP isokinetic dynomometer. These doses are dispensed in 3 x 118.3 mL aliquots of the 355 mL solution. Blood pressure is monitored 20 minutes pπor to testing. Once the final "zero mmute" dose of GAKIC or placebo is administered, the following fatigue testing protocol is initiated:
1 ) Immediately following the final administration of GAKIC or placebo (t = 0 mm), subjects perform 35 consecutive πght knee concentπc and eccentπc contractions using maximal effort at a speed of 90°/sec. The examiner gives verbal encouragement throughout each 35 repetition sequence. 2) Exactly 5 minutes following the final administration of GAKIC or placebo, subjects perform a second set of 35 consecutive πght knee concentric and eccentric contractions using maximal effort at a speed of 907sec. The examiner gives a verbal encouragement throughout each 35 repetition sequence.
3) Exactly 15 minutes following the third and final administration of GAKIC or placebo, subjects perform a third set of 35 consecutive πght knee concentπc and eccentπc contractions using maximal effort at a speed of 90° /sec. The examiner gives verbal encouragement throughout each 35 repetition sequence
4) Following this protocol and ample recovery time, subjects are dismissed and asked to return for testing after a 24 hour rest peπod. Subjects are allowed to eat or drink ad libitum, but are asked to document a wπtten dietary record of everything consumed for analysis of consumed L-leucme, L-argmme, glycme, total protein, caffeine, total calories, and total carbohydrate, as calculated using the Minnesota Nutrition Data software (version 2.91) developed by the Nutrition Coordination Center at the University of Minnesota.
Visit 3
Twenty four hours following the treatment, fatigue testing is performed as descπbed for visit 2, but is performed without delivery of GAKIC or placebo. Following 5 minutes of rest, the subject performs a set of 35 concentπc and eccentric knee extension repetitions at 90°/sec.
Subjects document a wπtten record of dietary intake for subsequent analysis of consumed L- leucine, L-argmme, glycine, total protein, caffeine, total calories, and total carbohydrate, as for
Visit 2. Subjects are instructed to return in 14 days for visit 4.
Visit 4
After 14 days, the same subjects crossover and repeat the protocol m a manner identical to Visit 2, with the assistant assigning and mixing the treatment which subjects did not receive in visit 2 (either 11.20 gm GAKIC glycine-arginme-α-ketoisocaproic acid-calcium salt, or 9.46 gm sucrose placebo m 355 ml Publix® Cranberry Juice) following another 12 hour fast with dietary recall records. During Visit 4, subjects again are seated on the Km-com 125AP isokinetic dynamometer and perform the same practice session descπbed in "Visit 2." Following 5 minutes of rest, subjects perform the set of 35 concentric and eccentric knee extension repetitions at 90° /sec. Subjects document a wπtten record of dietary intake for subsequent analysis of consumed L-leucme, L-argmme, glyc e, total protein, caffeine, total caloπes, and total carbohydrate, as for Visit 2. Subjects are instructed to return in 14 days for Visit 5.
Visit 5
Visit 5 is identical to visit 3. Twenty-four hours following the treatment, fatigue testmg is performed as descπbed for Visit 2, but is performed without delivery of GAKIC or placebo. Following 5 minutes of rest, the subject performs a set of 35 concentric and eccentric knee extension repetitions at 90°/sec. Data Collection/Analysis.
Measured torque is used as the dependent measure of force production. Torque produced duπng the last 5 repetitions of each 35 repetition testing set is used to calculate a mean torque value for the last five repetitions. The mean of these 5 repetitions is divided by the Baseline Maximal concentric or Baseline Maximal eccentric torque values determined duπng
"Visit 1." This creates a "Fatigue Resistance Index" (Figure 3) which represents the percentage of a subject's maximum ability to produce muscular force. Fatigue resistance reflects the subject's capability of sustaining dynamic muscular effort over a given time peπod. This ratio is obtained for each set m the protocol. A conceptual example comparing the "Fatigue Resistance Index" for two exercise sets is shown in Figure 3 for a case of 250 Newton-meters maximal baseline force from a single subject. Similar calculations are obtained for each subject at each visit as outlined m the Testing protocol overview (see Figure 2).
Total work for each set was measured. The values collected and derived from the Km- com software are in units of joules produced during each set. Differences in the values of Fatigue Resistance ratio and total work between the GAKIC trials and the placebo trials are measured using repeated measures ANOVA or paired t-tests. The withm-subjects group include treatment (GAKIC vs. placebo), and time following treatment oral administration (0, 5, 15 minutes, 24 hour). An a priori alpha significance level of 0.05 is used for all compaπsons.
Example 2 — Concentπc muscle fatigue resistance enhancement by GAKIC
Fasted subjects (N=13; repeated measures crossover) consumed oral GAKIC or isocaπc sucrose placebo, and then the concentric force excerted by their isolated right leg muscle (quadπceps femoπs) was measured following an exhaustive exercise regime controlled by the Km-com dynamometer. The concentπc Fatigue Resistance Index (FRI), shown m Figure 3, represented this force expressed as a percent of BIKEMSV. Greater FRI values indicated the ability of GAKIC treatment to sustain significantly greater muscle force (torque) and to significantly (p < 0.02) reduce acute muscle fatigue. Mean significant differences were sustained at 0 mm (50.2% vs. 39.2%), 5 mm (44% vs. 36.4%), and 15 mm (42.6% vs. 37.5%) for these same-subject repeated measures crossover paired comparisons, as shown m Figure 4. After 24 hr recovery, the treatment groups were not significantly different (P>0.05). The mean baseline concentπc isokinetic knee extension maximal torque was 241 ± 9 Nm. The concentπc FRI for each of the 35-repetιtιon sets obtained at 0, 5, and 15 mm following oral GAKIC treatment was greater (P < 0.02) than for isocaloπc carbohydrate (control). At these times, GAKIC treatment increased the mean FRI by 28%, 21%, and 13%, respectively, above lsocaloπc placebo control (carbohydrate) values. See Figure 5. These data indicated that GAKIC treatment increased the ability to maintain a higher percentages of the subjects' concentric baseline isokinetic knee extension maximum strength value (BIKEMSV) over these periods. After a 24 hour washout period, both GAKIC and placebo concentric forces were restored to the same absolute values with mean FRI=0.41 ± 0.05 (i.e., 98 N7240 N-m).
Analysis of values by order of treatment in the repeated measures cross-over design (i.e., GAKIC or placebo in either first or second set of trials) indicated no significant differences (P>0.05) at any time point, thus validating the random nature of the treatment assignment order.
Example 3 — Total work performed during each fatigue set for the concentric phase
GAKIC treatment means (±SEM) are significantly greater than placebo means (P<0.05) by repeated measures (N=13 subjects) comparisons for each 35-repetition set except after 24 hr recovery, when the treatment groups were not significantly different (P>0.05). See Figure 6.
For each 35-repetition fatigue set the difference in total concentric muscle work (joules) was obtained for placebo and GAKIC treatments. This Δ joules value represents the gain in concentric work directly attributable to GAKIC. The indicated points are significantly different (P<0.05) from (e.g., greater than) sucrose placebo group values treatment (N=13 repeated measures cross-over design). After 24 hour recovery, values were not significantly different (P>0.05). See Figure 7. The TW value is a measure of a subject's ability to sustain total work output across time.
TW values (joules) were obtained by integrating the work performed during every repetition in a given set for both the concentric and eccentric phases of contraction. TW values could be affected by changes in the absolute force peak level, changes in ability to sustain a given force peak level, or changes in magnitude of sustained force output during each repetition. During the 0, 5, and 15 min sets, GAKIC treatment resulted in greater concentric TW values compared to isocaloric carbohydrate treatment. At these times, GAKIC treatment increased the mean TW per set by 12%, 9%, and 11%, respectively, above isocaloric carbohydrate values. The overall mean percent gain in muscle total work attributable to G.AKIC treatment was 10.5 ± 0.8% retained for at least 15 min. See Figure 8. The 24 hr washout period restored the effect of GAKIC treatment to the control mean value of TW=4600 ±280 joules. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Claims

Claims
1. A method for enhancing muscle performance or recovery from fatigue wherein said method comprises administering a composition comprising a ketoacid and an amino acid wherein said ammo acid is cationic or dibasic.
2. The method, according to claim 1, wherein said composition further comprises a compound selected from the group consisting of glutamate, glutamine, and glycme.
3. The method, according to claim 2, wherein said composition comprises glycine.
4. The method, according to claim 1 , wherein said ketoacid is selected from the group consisting of α-ketoisocaproic acid, α-ketoisovaleπc acid, α-ketobetamethylvaleπc acid, pyruvic acid, and salts thereof.
5. The method, according to claim 4, wherein said composition comprises ╬▒- ketoisocaproic acid, or a salt thereof.
6. The method, accordmg to claim 1 , wherein said amino acid is selected from the group consisting of arginine, omithine, histidine, and lysine.
7. The method, according to claim 6, wherein said composition comprises arginine.
8. The method, according to claim 1, wherein said ammo acid and said ketoacid are conjugated.
9. The method, accordmg to claim 1, wherein said composition is administered orally.
10. The method, according to claim 9, wherein said composition is administered as a low caloπe beverage.
11. The method, accordmg to claim 1, wherein said composition further compπses cranberry juice.
12. The method, according to claim 1, wherein said composition is administered orally by capsules.
13. The method, according to claim 1, wherein said composition is administered intravenously.
14. The method, according to claim 1 , wherein said method results in an increase in the fatigue resistance index.
15. The method, according to claim 1, wherein said method increases the total work output.
16. The method, according to claim 1, wherein said method improves dynamic performance during concentric contraction.
17. The method, according to claim 1, wherein said method improves dynamic performance during eccentric contraction.
18. The method, according to claim 1, wherein said method is used on a human.
19. The method, according to claim 1, wherein said method is used on a horse.
20. A composition for enhancing muscle performance or recovery from fatigue wherein said composition comprises a ketoacid and an amino acid wherein said amino acid is cationic or dibasic.
21. The composition, according to claim 20, wherein said composition further comprises a compound selected from the group consisting of glutamate, glutamine, and glycine.
22. The composition, according to claim 21, wherein said composition comprises glycine.
23. The composition, according to claim 20, wherein said ketoacid is selected from the group consisting of ╬▒-ketoisocaproic acid, ╬▒-ketoisovaleric acid, ╬▒-ketobetamethylvaleric acid, pyruvic acid, and salts thereof.
24. The composition, according to claim 23, wherein said composition comprises ╬▒- ketoisocaproic acid, or a salt thereof.
25. The composition, according to claim 25, wherein said amino acid is selected from the group consisting of arginine, omithine, histidine, and lysine.
26. The composition, according to claim 20, wherein said composition comprises arginine.
27. The composition, according to claim 20, wherein said amino acid and said ketoacid are conjugated.
28. A method for objectively assessing and quantifying muscle work, fatigue and recovery of dynamic muscle function associated with acute anaerobic exercise wherein said method comprises establishing a Fatigue Resistance Index which is defined as pre-exhaustion torque divided by baseline peak torque.
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